Methods of preparation and composition of peptide constructs useful for treatment of autoimmune and transplant related host versus graft conditions

ABSTRACT

Peptide constructs including a first peptide segment which includes an amino acid sequence associated with autoimmune disease, asthma, allergy or xeno- or allograft transplantation rejections bonded directly or via a linker or spacer to a second peptide which binds to T cells and which will redirect the immune response from a harmful Th1 response to a less harmful Th2 response, or which will bind to T cells to initiate, but not complete, an immune response causing the T cells to which the first peptide binds, to undergo anergy and apoptosis, are useful in treating autoimmune conditions. For instance, the peptide construct NGQEEKAGVVSTGLIGGGDSAFDVLSFTAEEKAGVYK (SEQ ID NO:14) wherein Th2 stimulating Peptide G (SEQ ID NO:15) is covalently linked, via spacer GGG, to cardiac myosin molecule My1 (SEQ ID NO:16), can be used for treatment or prevention of myocarditis.

FIELD OF INVENTION

This invention relates to peptide constructs, i.e., polypeptidesobtained by linking together two or more peptides based on or derivedfrom different molecules, which are useful in the treatment orprevention of autoimmune diseases, asthma, allergies, and host versusgraft (or graft versus host) rejection, as well as to compositionscontaining same, methods for producing same and methods for using same.

BACKGROUND

Autoimmune conditions are characterized by the body attacking itself bymounting an immune response against itself. Various antigens often withdefined epitopes recognized for some HLA genotypes, have beenidentified, including those associated with insulin dependent diabetesmellitis (IDDM), rheumatoid arthritis (RA) [e.g., collagen type II390-402 IAGFKGEQGPKGE (SEQ ID NO:1), systemic lupus erythematousis(SLE), ankyosing spondylitis (AS), pemphius vulgaris (PV) [epidermalcell adhesion molecule desmoglein 190-204], multiple sclerosis (MS),Myelinproteolipid MPL [peptide sequence KNIVTPRT (SEQ ID NO:2],certaintypes of psoriasis, and uveoretintis (J. Hammer et al 1997, Adv.Immunol, 66:67; R. Tisch, et al, 1999, J. Immunol. 163:1178; J. Yoon, etal, 1999, Science 284:1183; P. J. Ruiz et al, 1999, J. Immunol.,162:3336; C. J. Krco, et al, 1999, J. Immunol. 163:1661). In othercases, peptides are known that induce in animals, a condition similar toones found in humans, such as GDKVSFFCKNKEKKC (SEQ ID NO:3) forantiphospholipid antibodies associated with thrombosis (A. Gharavi etal, 1999, J. Immunol., 163:2922) or Myelin peptides for experimentalautoimmune encephalitis as a model for MS (P. J. Ruiz, et al, 1999, J.Immunol., 162:3336; S. Araga, et al, 1999, J. Immunol., 163:476-482; N.Karin, et al, 1999, J. Immunol., 160:5188; L. M. Howard, et al, 1999, J.Clin. Invest., 103:281). Glutamic acid decarboxylase and specificpeptides have been identified for IDDM (R. Tisch, et al, 1999, J.Immunol., 163:1178; J. Yoon, et al, 1999, Science, 284:1183). Many ofthese conditions are also characterized by elevated levels of one ormore different cytokines and other effectors such as TNF (S. Kleinau, etal, 1999, J. Immunol. 162:4266; T. Preckel, et al, 1998, Eur. J.Immunol., 28:3706; P. Wooley, et al, 1992, J. Immunol., 151:6602) aswell as autoantibodies, including in some cases, anti-costimulatormolecules, in particular, those for CTLA-4 (CD152) on CD4+ cells (T.Matsui, et al, 1999, J. Immunol., 162:4328).

Efforts are underway to attack cells or cellular products of the immunesystem and thereby treat autoimmune conditions, allergies, asthma andtranplantation rejection using as reagents presumptive antigenicpeptides or proteins, peptides representing certain T cells, monoclonalantibodies, recombinant proteins binding various effector cells ormolecules such as TNF and IgE.

The following immunomodulatory approach contrasts with the mode ofaction for products which are antigen specific.

A fusion protein LFA-3TIP (Amevive™ from Biogen), purportedly a moleculecomposed of the first extracellular domain of LFA-3 fused to the hinge(CH2 and CH3 domains of human IgG1) which targets the CD2 receptor on Tcells, is being evaluated for psoriasis and for xeno- and allograftrejection. LFA-3TIP is bifunctional (i.e., two identical LFA-3regionsand TIP) and, therefore is a complex conjugate molecule.According to Biogen, LFA-3TIP is a recombinant fusion protein designedto modulate immune response by blocking the cellular pathway thatactivates T cells. It is presumed by the present inventor that thecompound is acting on a subset of memory effector cells with a downmodulation or re-direction of modulation activity.

These less antigen specific approaches also utilize monoclonalantibodies that act on activated T cells and down regulate them such asby anti-CD3 (Protein Design Laboratories) or block APC and T cellinteraction by anti-ICAM-3 (ICOS). MEDI-507 (Medimmune) is believed tobe a humanized monoclonal antibody, for psoriasis that also targets CD2,presumably for removing or inactivating those cell types. Otherdiseases, such as, tissue transplantation rejection and allergies arealso being tested by this approach. In contrast to acting on cellsurface markers, rhu-Mab-E25 (Genentech) is believed to be a humanizedmonoclonal antibody against IgE that binds to circulating IgE, with thegoal of preventing activation of mast cells. In contrast, otherresearchers are developing monoclonal antibodies to act on symptoms oragents directly causing disease symptoms. Remicade Infliximab (Centocor)is purported to be a monoclonal antibody to TNF. Anti CD40 ligand hasbeen used for treatment in animal model of MS (L. M. Howard, et al.,1999, J. Clin. Invest, 103:281). A recombinant generated designedprotein Enbrel (Immunex) is purported to comprise two molecules of r-DNAderived TNF receptor, and is intended to block TNF's action.

It should be noted, however, that many of these agents are notsufficiently disease specific and often recognize and could affectnormal cellular and body constituents that have a defined and necessaryrole in normal immune defenses which are still needed.

More antigen or disease specific approaches are exemplified by theattempt to treat MS patients by oral administration of myelin proteinswhich have recently been reported; the same group of searchers are alsousing collagen type II for treatment of patients with rheumatoidarthritis. These treatments are designed to attack at the level of thegut associated lymphoid tissues (GALT) to induce tolerance by antigenspecific suppression of the immune system. It is not known if thesetreatments use the intact protein or a hydrolyzate containing smallerpeptides. See D. Hafler, et al, 1988, J. Immunol., 141:181; K.Wucherpfennig, et al, 1990, Science, 248:1016; K. Ota, et al., 1990,Nature, 346;183; and H. Weiner, 1999, PNAS, 88:9161.

Several researchers are testing peptide based materials for treatment ofautoimmune conditions. One approach uses peptide as immunogen, givenorally in large quantities. The peptide represents a peptide sequencethat is thought to be the autoimmune epitope itself or a modified formwhich may also have altered binding or improved stability properties. Byuse of the peptide it is thought that either the normal peptide or analtered peptide ligand (APL) will bind to the T cell receptor (TCR) andinduce a state of anergy since the multiple sets of bindings that wouldoccur with antigen presentation with an antigen presenting cell (APC) donot occur (A. Faith, et al., 1999, J. Immunol., 162:1836; Soares, et al,1998, J. Immunol., 160:4768; M. Croft, et al, 1997, J. Immunol.,159:3257: L. Ding, et al., 1998, J. Immunol., 161:6614; and S. Hin, etal, 1999, J. Immunol., 163:2363). Some of the approaches with APLinclude using related amino acids such a D amino acids (U. Koch, et al,1998, J. Immunol., 161:421), amino acids with substituted side chains(R. De Palma, et al, 1999, J. Immunol., 162:1982), methylene groups toreplace peptide bonds in the peptide backbone (L. Meda, et al, 1996, J.Immunol., 157:1213) and N-hydroxyl peptides (S. Hin et al, J. Immunol.,163:2363). Several groups have studied the effect of varioussubstitutions of side chains and the MHC and TCR molecules (L.Raddrizani, et al, 1997, J. Immunol., 159:702; T. M. Clay, et al, 1999,J. Immunol., 162:1749; L. Radrizanni, et al., Eur. J. Immunol., 1999,29:660). With insulin activity it has been shown that a one amino acidchange on the β-chain can abolish its oral immune tolerance activity intwo (2) mechanistically different IDDM murine models (D. Homann, et al,1999, J. Immunol., 163:1833). While not an autoimmune epitope it hasbeen reported that a single change from threonine to alanine can abolishbiological activity (C. L. Sutherland, et al, 1999, J. Immunol.,162:4720); a switch from phenylalanine to alanine alters the bee venomphospholipase to an inactive form (A. Faith, et al, 1999, J. Immunol,162:1836); as does a switch from tyrosine to alanine change from activeto inactive for another system (S. Hausman, et al, 1999, J. Immunol.,162:5389).

In another approach based on peptide materials, truncated peptides ofautoimmune inducing epitope are used as antagonist in an animal model totreat the particular condition (N. Karin, et al, 1999, J. Immunol.,160:5188). Several groups are using synthetic amino acid polymers thatare considered to represent epitopes which contain Tyrosine (Y),Glutamic acid (E), alanine (A) and lysine (K) to target T cells such asCopolymer 1. In one study Copaxone is being used as an oral tolerancedelivery approach to treat MS patients. Copaxone is believed to be asynthetic copolymer of four amino acids (D. Hafler, et al, 1988,J.Immunol., 141:131). Modified peptides of peptide epitopes are reportedlybeing studies for treatment of various autoimmune conditions, includingMS and PV (desmoglein-3) (J. Hammer, et al, 1997, Adv. Immunol., 66:67;K. Wucherpfennig, et al, 1995, PNAS, 92:11935). The scientists doingthese studies may also be using Myelin proteolipid associated peptideepitope, a polymer or derivative of this epitope for MS, (Wucherpfennig,et al, Id.) .

Peptides that are unique to the T cell antigen receptor molecule arefound in a particular part of the variable region, usually the thirdhypervariable region of the beta chain of the T cell antigen Receptor(TCRβVX) (B. Kotzin, et al, 1991, PNAS, 88:9161; J. Oksenberg, et al,1990, Nature, 345:344; S. Zamil, et al, 1986, Nature, 324:258). One suchpeptide is apparently being evaluated for a psoriasis vaccine as IR 502and others for rheumatoid arthritis (D. P. Gold, et al, 1997, J.Neuroimmunology, 76:29). In this case an immune response to the TCRβV3(C. L. Sutherland, et al, 1999, J. Immunol., 162:4720) peptide isgenerated with the goal to have the body eliminate the particular Tcells and by so removing the T cells responsible for the condition,treating the condition. However, this approach has the potential ofeliminating other T cells that contain the same βV3 peptide sequencebesides the one responsible for the autoimmune condition.

Still another peptide approach uses complimentary peptide vaccine thatinduces T cell anergy and prevents Experimental Autoimmune Encephalitis(EAE) in rats by induction of anti-TCR antibodies (a la anti-idiotype)and thereby elimination of these cells (S. Araga, et al, 1999, J.Immunol., 163:476).

The present inventor has previously discovered and describedimmunomodulatory peptide constructs which include a first peptide whichis an antigenic peptide associated with disease or the causativeorganism of disease covalently bonded to a second peptide which is a Tcell binding ligand. These heterofunctional cellular immunologicalreagents are described in the commonly assigned U.S. Pat. No. 5,652,342,the disclosure of which is incorporated herein, in its entirety, byreference thereto. According to this patent, representative T cellbinding ligands include, for example, portions of MHC Classes I and II,such as, b-2-microglobulin, portions of LFA-3, portions of the Fc regionof the heavy chain of immunoglobulins, Ia⁺ molecules, Among the generaldisclosure of antigens associated with disease, mention is made ofantigens associated with auto-immunity, including diabetes, Rheumatoidarthritis and thyroiditis.

SUMMARY OF THE INVENTION

The present invention provides peptide constructs useful for treatmentof autoimmune disease, asthma, allergy, and tissue transplantationrejection (including both host-versus-graft and graft-versus-hostrejection), which differ from the above approaches used with antigenicpeptide alone. The novel constructs bind in an antigen specific mannerand redirect the T cell in the direction of a nondeleterious autoimmuneresponse, primarily from a Th1 to a Th2 immune response, but whereadvantageous, primarily from a Th2 to a Th1 immune response.Alternatively, the novel constructs include one peptide component whichwill bind to T cells associated with autoimmune disease, asthma,allergies or host versus graft or graft versus host rejection while asecond peptide component will bind to sites on the T cells which willpreclude the normal sequence of events required for cell activationthereby initiating an abortative T cell modulation resulting in cellanergy and apoptosis.

Specifically, the novel peptides of this invention include peptideconstructs of the following formula (I):P ₁ −x−P ₂   (I)

-   -   where P₁ is a peptide associated with autoimmune disease,        allergy or asthma, or tissue transplantation rejection and which        will bind to an antigen receptor on a set or subset of T cells;    -   P₂ is an immune response modifying peptide which will (i) cause        a directed immune response by said set or subset of T cells to        which the peptide P₁ is attached or (ii) bind to a T cell        receptor which will cause said set or subset of T cells to which        the peptide P₁ is attached to initiate, but not complete, an        immune response causing said set or subset of T cells to undergo        anergy and apoptosis; and    -   x is a direct bond or linker for covalently bonding P₁ and P₂.

The present invention also provides a first method for treating orpreventing inappropriate autoimmune response in individuals at risk forautoimmune disease, allergic reactions, asthma or host-graft orgraft-host rejection, wherein a pharmacologically effective amount of apeptide construct of formula (I) is administered to the individual toeffectively eliminate the set or subset of T cells involved in theautoimmune response.

The present invention also provides a second method for modulating aninappropriate autoimmune response in individuals at risk for autoimmunedisease, allergic reactions, asthma or host-graft or graft-hostrejection, wherein a pharmacologically effective amount of a, peptideconstruct of formula (I) is administered to the individual to redirectthe autoimmune response from a Th1 to a Th2 immune response, or from aTh2 to a Th1 immune response, whereby the inappropriate autoimmuneresponse is modulated to decrease or eliminate the adverse effectsassociated with the inappropriate autoimmune response.

DETAILED DESCRIPTION OF THE INVENTION

It has been reported that the, “failure of mature CD8 cells tosimultaneously engage their TCR and CD8 coreceptor triggers anactivation process that begins with inhibition of CD8 gene expressionthrough remethylation and concludes with up-regulation of surface fasand fas ligand and cellular apoptosis” (G. A. Pestano, et al, 1999,Science, 284:1187). This is consistent with the results of others whereif full engagement of certain very major coreceptors are not effectedthen an activation process, but abortative in nature, leading toapoptosis occurs (see also P. Gogolak, et al, 1996, Immunol. Let.54:135; Grakoui, et al, 1999, Science, 284:221; Malissen, 1999, Science,285:207; S. Redpath, et al. 1999, J. Immunol., 163:6; T. Preckel, et al,1998, Eur. J. Immunol., 28:3706; S. Sambhara, et al, 1991, Science,252:1424; H. Kishimoto, et al, 1999, J. Immunol., 163:1817; Kubo, et al,1999, J. Immunol., 163:2432). Therefore, a different approach would beto have a modulation but not with a full sequence of events, theconstruct binding in an antigen specific manner with the antigenicepitope but the TCBL ligand binding to a site on another moleculeassociated with certain early events that are early intermediates in thefull expression pathway thereby occupying the space and causing an earlyevent in the process of activation (such as Ca⁺⁺ flux, activation ofvarious phosphatases, membrane migration events, such as “patching” or“capping”, changes in RNA metabolism) but not supporting the completeactivation process which can be thought of as culminating by antigenspecific non-antibody mediated Cytotoxic T Lymphocyte activity such askilling of infected or tumor cells, DNA synthesis and cell division, andcytokine secretion, namely, without allowing the ultimate tertiarycomplex of binding events (MHC, antigen TCR and CD4 (or CD8)) necessaryfor full activation by being out of the normal temporal sequence ofevents. Perhaps this early binding would be of such strength that itdoes not disassociate and allow the cell surface rearrangement necessaryfor the full and normal sequence of modulatory events, such as,proliferation or secretion of late cytokines such as Fas, TNF-α or IFN-γand thereby prohibiting events found in an autoimmune disease associatedpathway with complete T cell activation. For example, initially afterantigen binding to the TCR ICAM-1 (also known as CD54) on APC binding toa T cell's LFA-1 (also known as CD11a/CD18) and then is shifted away anda rearrangement with clustering of the MHC and antigenic peptides on APCbinding eventually by migration on T cell membrane to a clustering of TCand CD4 (or CD8) (B. Malissen, 1999, Science, 285:207; A. Grakoui, etal, 1999, Science, 285:221).

According to one embodiment of this invention, such rearrangement isprevented by the close association in a peptide construct using a TCBLfrom ICAM-1, LFA-3 (aa26-42), VLWKKQKDKVAELENSE (SEQ ID NO:4) (L.Osborn, et al, 1995,JEM, 181:429), by either the disparity in thetemporal binding or higher strength of binding activity, therebypreventing the rearrangements and other more intimate interactionsnecessary for activation. Initially these sites are close together butnormally rearrangements on the T cell surface occur during theactivation process so by preventing this shift activation should notoccur. Likewise, a TCBL from CD4 that binds to the TCR and CD3 may beused as the TCBL in the peptide construct of this invention. Its bindingto the T cell recognition site will inhibit subsequent events fromoccurring (MHC II with CD4 or β-2 with CD8).

Still another approach is a construct which redirects the immuneresponse initiated by the natural autoimmune inducing event from a TH1to a TH2 response (see, e.g., Lowrie, et al, 1999, Nature, 400:269; R.Tisch, et al, 1999, J. Immunol., 163:1178). As used herein, a TH2directed response is one which directs the immune response toward theTH2 direction, thus favoring production of more IL-5, IL-4,TNF-α-cytokines and antibody isotypes IgG1 and IgG3 in mice (orcomparables in man) as opposed to Th1, where the immune response favorsproduction of IFN-γ, IL-2, IL-6, IL-12 cytokines and antibody isotypesIgG2a and IgG2b in mice and Cytotoxic T cell activity. It is understood,of course, that a “TH2 directed response” is not intended to imply anexclusively TH2 response, but rather a mixed immune response which isweighted to favor a TH2 profile.

According to this embodiment a TCBL associated with TH2 responses; e.g.,peptide G from MHC class II (D. Zimmerman, et al, 1996, Vacc. Res.,5:91, 5:102; K. Rosenthal, et al, 1999, Vaccine), IL-4 or IL-5 orpeptides known to stimulate Il-4 or IL-5 synthesis are used as the TCBLalong with the autoimmune inducing peptide (see, e.g., Hammer, et al,Krco, etal, Araga, et al, Ota, et al, Ruiz, et al, Yoon, et al, supra,Dittel, et al, 1999, J. Immunol., 163:32; Gautam, et al, 1998, J.Immunol., 161:60, the disclosures of which are incorporated herein byreference thereto) in the peptide conjugate. These peptide constructsmay be used, for example, to treat type I diabetes. In an animal modelthe mechanism of diabetes prevention in the RIP-NP model was shown to bemediated by insulin β-chain, and Il-4 producing regulatory cells actingas bystander suppressors (D. Homann, et al, 1999, J. Immunol.,163:1833). Such redirection of immune responses have been previouslyreported by a DNA vaccine for TB which redirected the immune responsefrom an inefficient response TH2 to a response that was a very effectiveTh1 (Lowrie, et al, 1999, Nature, 400:269). Thus, redirecting an alreadyexisting immune response from a TH1 to a TH2 would be effective fortreating autoimmune related diseases. A TCBL involved in CD28costimulation (Kubo, et al, supra) could also be effective for thispurpose. If, on the otherhand, the need was to redirect from a TH2 to aTH1, much less likely to be needed since many autoimmune conditions arethought to be the manifestation of deleterious TH1 effects, then a TCBLsuch as peptide J DLLKNGERIEKVE (SEQ ID NO:51) (D. Zimmerman, et al,supra; K. Rosenthal, et al, supra) or ones known to stimulate IL-2 orIL-12 synthesis, would be used along with the autoimmune inducingpeptide.

Yet another approach is to use the peptide construct to not activate thenormal immune process but to activate the process leading to apoptosisof the T cell by using as the TCBL a ligand that binds to a site on theT cell whose normal binding and activation leads to apoptosis of the Tcell; such as the TNF-receptor of the T cell, in which the TCBL would bethe TNF-α ligand portion. Examples of such TNF peptides known toactivate macrophages are amino acids 70-80 PSTHVLITHTI (SEQ ID NO:5) (W.J. Britton, et al, 1998, I & I, 66:2122) and perhaps the antagonistpeptide represented by DFLPHYKNTSLGHRP (SEQ ID NO:6) of another region(C. Chirinos-Rojas, et al, 1998, J. Immunol., 161:5621). It has beensuggested in WO 99/36903A1 that the H4-1-BB Ligand is useful as atreatment for autoimmune disease similar to uses for flt3-L and CD40L;therefore, H4-1BB may also be used as TCBL for inclusion with autoimmuneantigens to form the inventive peptide construct. Other such TCBLexamples are available from application with Fas and Fas-ligandincluding the noncleavable Fas-ligand (WO 99/36079A1).

Since autoimmune reactive cells in disease are most likely alreadyactivated and may be expressing Fas (Y. Tomita, et al, 1996, Int. J.Can., 68:132; B. Lie, et al, 1996, Biol. Pharma. Bul. 19:1602) theFas-Ligand or the sequence obtained by reverse engineering technique todetermine amino acid (aa) sequence acting as receptor for DEVD-aldehydeor YVAD chloromethylketone, may also be used as the TCBL.Representatives from another pair, IFN-γ and the IFN-γ ligand can alsobe used as TCBLs in the invention peptide constructs.

In this invention the antigenic peptide and the peptide for T cellbinding (TCBL) may be directly linked together in any order. (i.e.,N-terminal of one to C-terminal of other or vice versa) or the peptidemay be covalently bonded by a spacer or linker molecule. With regard tolinkers between the two domains, suitable examples include a thioetherbond between an amino terminus bromoacetylated peptide and a carboxylterminus cysteine, often preceded by a diglycine sequence (D. Zimmerman,et al, supra), carbodiimide linkages, a multiple glycine, e.g., from.3to 6 glycines, such as triglycine, with or without one or two serines,separation between the two entities, e.g., GGGS (SEQ ID NO:7), GGGSS(SEQ ID NO.8), GGGGS (SEQ ID NO:9), GGGGSS (SEQ ID NO.10), GGGSGGGS (SEQID NO:11), etc., and other conventional linkages, such as, for example,the direct linkages such as, EDS, SPDP, and MBS, as disclosed in theaforementioned U.S. Pat. No. 5,652,342.

Thus, the peptide constructs of this invention may be convenientlyrepresented by the following formula (I):P ₁ −x−P ₂   (I)

-   -   where P₁ is a peptide associated with autoimmune disease,        allergy or asthma, or transplantation rejection and which will        bind to an antigen receptor on a set or subset of T cells;    -   P₂ is an immune response modifying peptide which will bind to T        cells to cause a directed immune response by said set or subset        of T cells to which the peptide P₁ is attached or which will        bind to a T cell receptor which will cause said set or subset of        T cells to which the peptide P₁ is attached to initiate, but not        complete, an immune response causing said set or subset of T        cells to undergo anergy and apoptosis; and    -   x is a direct bond or linker for covalently bonding P₁ and P₂.

The TCBL portion of the immunomodulatory peptide construct of thisinvention, i.e., P₂, may comprise a discontinuous epitope composed oftwo small regions separated by a loop or by a single chain short peptidein place of the loop (D. Shan, et al, 1999, J. Immunol., 162:6589). Forexample, an eight amino acid group, LRGGGGSS (SEQ ID NO:12), of 11.2Angstroms in length (U. Reineke, et al, 1999, Nature Biotechnology,17:271) has been used to form a single peptide from two smallerdiscontinuous peptides of IL-10, thereby forming a TCBL which could beused for redirection from a TH1 to a TH2, in combination with, forexample, the IDDM, PV or MS inducing epitopes (see Hammer, et al, supra,Tisch, et al, supra). Linkers of varying lengths to form a single chainmay be used, for example, GGGS (SEQ ID NO:7), GGGGS (SEQ ID NO:9),including, from among 1 or more repeats of this tetrapeptide orpentapeptide, e.g., GGGSGGGS (SEQ ID NO:11), GGGSGTGSGSGS (SEQ ID NO:52)(See, Uger, et al. The J. of Immunology, 162:6024-6028, 1999) etc. Suchlinkers may result in a tertiary structure which might be of use to forma more avid TCBL (see D. Shan, et al, supra).

Although not strictly limited, the peptide constructs of this inventionmay have as many as about 200 amino acids in its sequence, preferably upto about 150 amino acids, and especially, up to about 100 amino acids.The minimum number of amino acids is also not strictly limited butusually each of the peptide components P₁ and P₂ will have at leastabout 4, preferably at least about 6, and more preferably at least about8 or 9 amino acids in order to provide the appropriate epitopeconfiguration for effectively binding to the appropriate site on the Tcells of interest. Thus, the peptide constructs of this invention willusually contain from about 20 to about 100 or more amino acids.

The peptide constructs may be prepared using conventional solid statepeptide synthesis, provided however, that for constructs having morethan about 40 amino acids, especially more than about 50 amino acids, itis usually convenient and preferred to prepare shorter segments and thenlink the shorter segments using well known techniques in solid phasepeptide synthesis.

Alternatively, the peptide constructs of this invention may be preparedusing well known genetic engineering methods.

Further details on methods for producing the instant peptide constructscan be gleaned from the aforementioned U.S. Pat. No. 5,652,342.

The novel peptide constructs of this invention can also be used to treattransplant recipients, for example, for heart, liver, lung and kidney,who are either undergoing or at risk for rejection of the transplant(Host versus Graft or HvG). In this case, the peptide antigen(s) used tomake the construct would be the major epitope(s) of the graft combinedwith a TCBL as described above to either suppress or redirect the immuneresponse. As one of the major sources of antigenic materials causing theHvG are thought to be the MHC proteins and, in particular, thepolymorphic regions that are from the graft tissue and recognized by thehost's MHC, peptides from those regions of the graft's MHC moleculeswould be likely candidates for the antigenic epitope portion. Otherregions of potential usefulness include polymorphic regions of the minorhistocompatability antigens. TCBLs can be selected from, but are notrestricted to the following LFA-3 or FasL described above (see, B. Lie,et al or Y. Tomita, et al, supra). Peptide G (SEQ ID NO:15) from the MHCII molecule, or Hu IL-10 (SEQ ID NO:28) (B. Gesser, et al, 1998, PNAS,94:14620; Lie, et al, supra, and Y. Tomita, et al, supra) may beselected for redirection of immune responses.

As activated T cells normally express MHC molecules, another way ofimmunomodulation is to take advantage of the programmed pathwayestablished by antigen addition. T cells which receive a signal from theTCR and the MHC I to CD-8⁺ cells undergo apoptosis without othercostimulatory signals (S. Sambhara, et al, 1991, Science, 252:1424).Therefore, the TCBL, peptide E, (the α3 domain amino acids 223-229 ofthe human MHC I conserved region can be used along with the autoimmuneepitope to form a peptide construct according to another embodiment ofthis invention.

The peptide constructs of this invention may be used as or in vaccinesas therapeutic agents for treatment of autoimmune disease or HvG. Thevaccines will be administered often but not always with an adjuvant andon a regular regimen such as weekly, biweekly, monthly, quarterly,semi-annually or annually by one of the following routes, ID, IM orSub-cu and perhaps also as a cutaneous transdermal or nasal deliveryvehicle in amounts of from 1-100, usually 10-50, micrograms per kilogramof body weight.

If a binding site is known on the target T cell with a defined aminoacid sequence and the TCBL amino acid sequence on the ligand is notknown then determination of the DNA encoding for the peptide would allowfor determination of the cDNA and thus the complementary peptidesequence using the technique of Lie, et al, supra, for example.

In general, the TCBL's (P2) used to form the peptide constructs of thisinvention will be selected from those for normal induction andmodulation of immune responses, including those selected to effectredirection from Th1 or Th2, including, for example, those that areknown to be related and involved in the normal events of activation,namely, IL-2, IL-10, IL-12, IL-4, IL-1β 163-171 (VQGEESNDK (SEQ IDNO:13)) (see, e.g., K. Bajpai, et al, 1998 Immunopharmacology, 38:237;W. Beckers, et al, 1993, J. Immunol., 151:1757; D. Fresca, et al, 1988,J. Immunol., 141:2651; G. Antoni, et al, 1986, J. Immunol, 187:3201),and most likely derived from the final complexes (MHC-I or II and CD8 orCD4) TCR and antigenic peptide. Examples of TCBLs that are associatedwith earlier activation events, include for example, Fas and FasL, TNF-αand TNF-αR and those for formation of early intermediate complexes andLFA-3 and CD2.

Examples of antigens associated with autoimmune disease, allergy,asthma, and transplantation rejection, include, for example, thosementioned above, including the Background discussion and those shown inthe following, non-limitating, representative examples, as well as inthe literature references cited herein, the disclosures of which areincorporated herein, in their entirety, by reference thereto. Additionalexamples, may also be found in the following literature, C. de Lalla, etal, 1999, J. Immunol., 163:1725 (Lol p5a allergen from rye grass); A. M.Gautam, et al, 1998, J. Immunol., 161:60 (experimental autoimmuneencehpalomyelitis); as well as many others available to thepractitioner.

The invention will now be described in further detail by way ofrepresentative examples, however, the present invention is not limitedto the examples and should be construed to cover the subject matter asdefined in appended claims and equivalents thereto.

EXAMPLE 1

A conjugated peptide NGQEEKAGVVSTGLIGGGDSAFDVLSFTAEEKAGVYK (SEQ ID NO:14) is obtained by covalently bonding Peptide G (NGQEEKAGVVSTGLI (SEQ IDNO:15)) with the cardiac myosin molecule My1 (DSAFDVLSFTAEEKAGVYK (SEQID NO:16)) via a GGG spacer. This peptide construct

NGQEEKAGVVSTGLIGGGDSAFDVLSFTAEEKAGVYK (SEQ ID NO:14) can be used in avaccine for treatment or prevention of myocarditis.

EXAMPLE 2

Addison's disease is characterized with an immune reation to the enzymesteroid 21 hydroxylase (21OH) with implication of a conformationalepitope composed of peptides from the regions at 447-461 and 471-491(see Nikoshkov et al, 1999, J. Immunol. 162:2422). Accordingly, thefollowing peptide construct would be useful in the treatment ofAddison's disease and combines the conformational epitope peptides fromaa447-461 and aa474-491 of the enzyme steroid 21 hydroxylase (21OH) as asingle chain linked with LRGGGGSS (SEQ ID NO:12) and the linked peptidesare bounded via a GGGGS (SEQ ID NO:9) linker with a LFA-3 peptide (e.g.,VLWKKQKDKVAELENSE (SEQ ID NO:4), aa 26-42 (see L. Osborn et al, J. Exp.Med. 181:429). Accordingly, the peptide construct will have thefollowing formula:VLWKKQKDKVAELENSE-GGGGS-21OH₄₄₇₋₄₆₁-LRGGGGSS-21OH₄₇₇₋₄₉₁ (SEQ ID NO:17)LFA-3₂₆₋₄₂     linker 21OH₄₄₇₋₄₆₁  linker  21OH₄₇₇₋₄₉₁

EXAMPLE 3

The following construct will be useful in the treatment of myastheniagravis: A TCBL from IL-10, aa.₁₅₂₋₁₆₀ AYMTMKIRN (SEQ ID NO:18) (seeGesser, et al, 1997, Proc. Nat. Acad. Sci. 94:14620) is linked to anepitope associated with acetylcholine receptor (AchR), EIIVTHFPFDEQNCSMK(SEQ ID NO:19), aa129-145 (see, H. Yoshikawa et al 1997 J. Immunol159:1570) to form (SEQ ID NO:20) AYMTMKIRN-GGGGS-EIIVTHFPFDEQNCSMKHu-IL-10₁₅₂₋₁₆₀linker    AchR₁₂₉₋₁₄₅

Similar results can be obtained by using as the TCBL, in place ofHu-IL₁₅₂₋₁₆₀, the MHC I α3 domain peptide 223-229 (DQTQDTE (SEQ IDNO:21)) that binds to the CD8 molecule, to form the following construct:DQTQDTE-GGGGS-EIIVTHFPFDEQNCSMK (SEQ ID NO: 22)MHC-1α₂₂₃₋₂₂₉linker   AchR₁₂₉₋₁₄₅

EXAMPLE 4

Rheumatoid arthritis (RA), is a condition where excess Tumor necrosisfactor-α (TNF-α) production is a problem and a major point of attack fornew treatments. The following peptide construct is prepared for use inthe treatment of RA: PSTHVLITHTI-GGG-IAGFKGEQGPKGE (SEQ ID NO:23)TNF-α₇₀₋₈₀  linker    C-II₃₉₀₋₄₀₂where the peptide TNF-α₇₀₋₈₀ is known to activate macrophages (10) andthe collagen type II peptide C-II₃₉₀₋₄₀₂ (IAGFKGEQGPKGE (SEQ ID NO:40))is an epitope associated with RA. Thus, this peptide construct will beuseful to achieve abortative T cell modulation since the impropersequence of events occurs with a binding that precludes the normalactivation process.

Similar effects will be achieved using a peptide construct similar tothe above but obtained by using, in place of TNF-α peptide α₇₀₋₈₀, thefollowing: TNF-α antagonist: DFLPHYKNTSLGHRP (SEQ ID NO:24) (see C.Chirinos-Rojas et al, J. Immunol 161:5621), namely, (SEQ ID NO:25)DFLPHYKNTSLGHRP-GGG-IAGFKGEQGPKGETNF-α_(antagonist peptide) spacer  C-II₃₉₀₋₄₀₂

EXAMPLE 5

As a different approach for treating RA the following peptide constructis prepared utilizing peptide G or a derivative thereof (derG)(DGQEEKAGVVSTGLI (SEQ ID NO:50)) from MHC-IIβ2(135-149) to redirect theimmune response from a TH1 to a TH2: NGQEEKAGVVSTGLI-GGGGS-IAGFKGEQGPKGE(SEQ ID NO:26) G(MHC-IIβ2)    linker    C-II₃₉₀₋₄₀₂ orDGQEEKAGVVSTGLI-GGGGS-IAGFKGEQGPKG (SEQ ID NO:27)derG(MHC-IIβ2)  linker   C-II₃₉₀₋₄₀₂

EXAMPLE 6

Another example of a peptide construct according to this invention forredirecting the immune response from a TH1 to a TH2, is used in thetreatment of autoimmune myocarditis is obtained by linking as a TCBL thesingle chain construct (see D. Shan et al, 1999, J. Immunol., 162:6589)using the discontinuous regions of Hu IL-10 having the sequenceDNQLLETCKQDRLRNRRGNGSSTHFEGNLPC (SEQ ID NO:28) with the cysteines at aa8and 31 cyclized in a disulfide bond ring (see, e.g., U. Reinke et al,1999, Nature Biotechnology, 17:271) linked via a GGGGS (SEQ ID NO:9)spacer to an autoimmune epitope from My-1 from murine cardiac myosin(see D. L. Donermeyer, et al, 1995, J. Exp. Med., 182:1291) or the humancounterpart: DNQLLETCKQDRLRNRRGNGSSTHFEGNLPC-GGGGS-DSAFDVLSFTAEEKAGVYK(SEQ ID NO:29) Hu IL-10 discontinuous epitopes linker        My-I₃₃₄₋₃₅₂

EXAMPLE 7

This example shows a peptide construct for treating autoimmune responseassociated with the myelinproteolipid protein (MPLP) having the sequenceVHFFKNIVTPRTP (SEQ ID NO:41) using the MHC-Iα₂₂₃₋₂₂₉ TCBL:DQTQDTE-GGGGSS-VHFFKNIVTPRTP (SEQ ID NO:30) MHC     linker    MPLP-Iα₂₂₃₋₂₂₉

EXAMPLE 8

This example illustrates an embodiment of a peptide construct useful intreating an autoimmune disease, thrombosis, by allowing initiation of animmune response but not allowing full development and completion.

In particular, the peptide GDKVSFFCKNKEKKC (SEQ ID NO:3) forantiphospholipid (PL) antibodies associated with thrombosis (seeGharavi, et al, 1999, J. Immunol. 163:2922) is linked to a MHC-I TCBL aspreviously described: DQTQDTE-GGGGSS-GDKVSFFCKNKEKKC (SEQ ID NO:31)MHC-Iα₂₂₃₋₂₂₉ linker    PL

EXAMPLE 9

This example illustrates a peptide construct for redirecting the immuneresponse from IgE to another more benign immune response. Similarpeptide constructs to that shown below would be useful to treat variousallergic conditions resulting from allergy epitopes, including, forexample, pollens, such as from grasses and trees; venoms, such as frombee venom; danders such as from cow, cat, dog, rabbit, mouse, rat, andother household pets; and dust mite antigens, simply by replacing theexemplified Bos d2 allergen peptide with the appropriate allergenpeptide. DQTQDTE-GGGGSS-YQQLNSERGVPNENIEN (SEQ ID NO:32)MHC-Iα₂₂₃₋₂₂₉ linker  Bos d2₁₃₁₋₁₄₈where Bos d2₁₃₁₋₁₄₈ (YQQLNSERGVPNENIEN (SEQ ID NO:42)) is the cow danderallergen of Lipocalin, epitope “G” at carboxyl terminus (T. Zeiler et al1999, J. Immunol. 162:1415).

EXAMPLE 10

Another approach to a peptide conjugate for treating allergic conditionsis achieved with the following peptide conjugate which will initiaterecognition and the activation process but without allowingreorientation and, therefore, will cause truncation of the immuneresponse with premature cessation, resulting in cell anergy andapoptosis. (SEQ ID NO.33) VLWKKQKDKVAELENSE-GGGGSS-IQTQMKTYSDIDGKLVSEV   LFA-3₂₆₋₄₂      linker    ParJ1₄₇₋₆₅where ParJ1₄₇₋₆₅ (IQTQMKTYSDIDGKLVSEV (SEQ ID NO:43)) is a pollenantigen from the common plant from the mediterian region of Parietariajudaaica (R. De Palme et al J. Immunol. 162:1836).

EXAMPLE 11

Similarly to the peptide construct of Example 10, the following peptideconstructs would similarly cause T cell activation followed by prematurecessation of the immune response: (SEQ ID NO:34)VLWKKQKDKVAELENSE-GGGGSS-VFIKRVSNVIIHG    LFA-3₂₆₋₄₂       linker   Cry j1₁₀₈₋₁₂₀

where Cry j1₄₇₋₆₅ (VFIKRVSNVIIHG (SEQ ID NO:44)) is the Japanese cedarpollen antigen from Cryptomeria Japonica, aa47-65; (SEQ ID NO:35)VLWKKQKDKVAELENSE-GGGGSS-MKVTVAFNQFGPNRR   LFA-3₂₆₋₄₂       linker   Cry j1₁₁₂₋₂₂₅

where Cry j1₂₁₁₋₂₂₅ (MKVTVAFNQFGPNRR (SEQ ID NO:45)) is the Japanesecedar pollen antigen from Cryptomeria Japonica, aa221-225; (SEQ IDNO:36) VLWKKQKDKVAELENSE-GGGGSS-IASRRVDGIIAAYQN  LFA-3₂₆₋₄₂        linker    Cry j2₆₆₋₈₀

where Cry j2₆₆₋₈₀ (IASRRVDGIIAAYQN (SEQ ID NO:46)) is the Japanese cedarpollen antigen from Cryptomeria Japonica, Cry j2, aa66-80; (SEQ IDNO:37) VLWKKQKDKVAELENSE-GGGGSS-IDIFASKNFHLQKNTIGTG  LFA-3₂₆₋₄₂        linker   Cry j2₁₈₂₋₂₀₀where Cry j2₁₈₂₋₂₀₀ (IDIFASKNFHLQTIGTG (SEQ ID NO:47)) is the Japanesecedar pollen antigen from Cryptomeria Japonica, Cry j2, aa182-200 (see,e.g., T. Sone, et al, 1998, J. Immunol., 161:448).

Similarly, by replacing the allergen antigen in the above conjugatedpeptides with, for example, the allergen antigen from other plants,grasses and trees, other conjugated peptides effective in the treatmentof the associated allergic condition will be obtained. Examples of suchother allergen antigens include olive tree Olea europea pollen (locatedin a 17 kDa protein, P. Lauzurica et al, 1988, Mol. Immunol. 25:329);rye grass pollen Lolium perenne, major epitope shared between Lol p I,II, III (Ansari, et al, 1987, J. Immunol. 139:4034) and Lol p 5a₁₄₅₋₁₇₀(Schramm et al, 1999, J. Immunol. 162:2406); Timothy grass pollen rPh1p1, p2, p5₈₅₋₁₂₀ and P5₂₁₀₋₂₄₀.

EXAMPLE 12

Other peptide constructs useful in the treatment of allergic conditionsassociated with dust mite antigens, are prepared by linking. TCBLs, suchas LFA-3 or MHC-1α, with, for example, fecal allergen of miteDermatophagoides pteronyssimus Der p1 and related mites, known to inducean IgE reaction (Lind et al 1988 J. Immunol. 140:4256; Platts-Miils etal 1987 J. Allerg. Clin. Immunol 80:755), but redirected in accordancewith this invention. Moreover, since it is known that Der p1 and Der p9trigtger release of GmCSF, IL-6, IL-8, etc. (C. King et al 1998 J.Immunol. 161:3645), these constructs with LFA-3 or MHC-1α would beexpected to be of benefit by the activation/truncation mechanism leadingto cell anergy and apoptosis as previously described.

EXAMPLE 13

(SEQ ID NO:38) VLWKKQKDKVAELENSE-GGGGSS-YFVGKMYFNLID LFA-3₂₆₋₄₂      linker   PLA A2₈₁₋₉₂

where PLA A2₈₁₋₉₂ (YFVGKYFNLID (SEQ ID NO:48)) is a bee venom antigen(A. Faith et al 1999 J. Immunol 162:1836) or (SEQ ID NO:39)DQTQDTE-GGGGSS-GIGAVLKVLTTGPALISWIKRKRQQ  MHC-Iα    linker      Melittinwhere Melittin (GIGAVLKVLTTGPALISWIKRKRQQ (SEQ ID NO:49)) is a Honey Beeantigen which has been shown to induce IgE in about ⅓ of bee allergicpersons (T. P. King et al 1994, J. Immunol. 153:1124); are peptideconstructs according to the invention which can be administered to beeallergic persons to protect such treated individuals against allergicreactions resulting from bee stings.

1. An immunomodulatory peptide construct having the formula P ₁ −x−P ₂ where P₁ is a peptide associated with autoimmune disease, allergy or asthma, or host-versus-graft rejection and which will bind to an antigen receptor on a set or subset of T cells; P₂ is an immune modulating peptide which will (i) cause a directed immune response by said set or subset of T cells to which the peptide P₁ is attached or (ii) bind to a T cell receptor which will cause said set or subset of T cells to which the peptide P₁ is attached to initiate, but not complete, an immune response causing said set or subset of T cells to undergo anergy and apoptosis; and x is a direct bond or linker for covalently bonding P₁ and P₂.
 2. The immunomodulatory peptide construct according to claim 1, wherein peptide P₁ is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49.
 3. The immunomodulatory peptide construct according to claim 1, wherein peptide P₂ is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:50, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:28 and SEQ ID NO:51.
 4. The immunomodulatory peptide construct according to claim 1, which is selected from the group consisting of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39.
 5. The immunomodulatory peptide construct according to claim 1, wherein x represents a linker comprising a sequence having from 3 to 6 glycine residues and from 0 to 2 serine residues.
 6. A method for eliminating a set or subset of T cells involved in autoimmune response which comprises administering to an individual at risk for autoimmune disease, allergic reactions, asthma or host-graft or graft-host rejection, a pharmacologically effective amount of a peptide construct of formula (I), as set forth in claim 1, whereby, the autoimmune response may be totally or substantially prevented.
 7. A method for interrupting an autoimmune disease associate pathway necessary to complete T cell activation, comprising administering to an individual at risk for autoimmune disease, allergic reactions, asthma or host-graft or graft-host rejection, comprising administering to said individual, a pharmacologically effective amount of a peptide construct of formula (I) as set forth in claim 1, whereby the antigenic peptide P₁ comprises an epitope binding to a the surface of a T cell in a set of T cells or subset of T cells, in an antigen specific manner, and wherein the peptide P₂ comprises an epitope which will bind to a T cell receptor site on the surface of said T cell which will cause said set or subset of T cells to initiate, but not complete, an immune response to thereby cause said set or subset of T cells to undergo anergy and apoptosis.
 8. The method according to claim 7, wherein the peptide P₂comprises the peptide having SEQ ID NO:4.
 9. The method according to claim 7, wherein the peptide P₂ comprises a peptide selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6.
 10. A method for modulating an inappropriate immune response in an individual at risk for autoimmune disease, allergic reactions, asthma or host-graft or graft-host rejection, comprising administering to said individual a pharmacologically effective amount of a peptide construct of formula (I), as set forth in claim 1, whereby the autoimmune response is redirected from a Th1 to a Th2 immune response, thereby modulating the inappropriate immune response to decrease or eliminate the adverse effects associated with the inappropriate autoimmune response.
 11. The method of claim 10, wherein the peptide P₂ comprises a peptide selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:50.
 12. A method for modulating an inappropriate immune response in an individual at risk for autoimmune disease, allergic reactions, asthma or host-graft or graft-host rejection, comprising administering to said individual a pharmacologically effective amount of a peptide construct of formula (I), as set forth in claim 1, whereby the autoimmune response is redirected from a Th2 to a Th1 immune response, thereby modulating the inappropriate immune response to decrease or eliminate the adverse effects associated with the inappropriate autoimmune response.
 13. The method of claim 12, wherein the peptide P₂ comprises the peptide having SEQ ID NO:21 or SEQ ID NO:51. 